Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

Article abstract of DOI:10.1021/jo051836t

The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding wi

Full text of DOI:10.1021/jo051836t

Design of New Bidentate Ligands Constructed of Two Hoechst
33258 Units for Discrimination of the Length of Two A3T3 Binding
Motifs
Mikimasa Tanada, Saori Tsujita, and Shigeki Sasaki*
Graduate School of Pharmaceutical Sciences, Kyushu UniVersity, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
sasaki@phar.kyushu-u.ac.jp
ReceiVed August 31, 2005
The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs
cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258
units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is
introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA
binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular
dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1
with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H
(1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated
by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way
junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs
can be selectively recognized by the newly designed bidentate ligands.
Introduction
that not only the sequences but also higher-ordered structures
of DNA or RNA play significant roles in regulation of gene
expression,8-10 and therefore increasing attention has been paid
to development of small molecular ligands for targeting such
structures. Triplex-binding ligands are used in antigene approach
for inhibition of transcription,11-13 and telomerase activity is
inhibited by quadruplex-binding ligands.14-16 Quadruplex-
binding ligands were also used to detect G-quartet structure in
Molecules that bind duplex DNA sequence selectively have
been of great interest because of potential applications in
genomic studies as well as therapeutic purposes.1-5 Small
molecules are of special concern because they are easily
accessible to chromosomal DNA.6,7 Recent studies have shown
* To whom correspondence should be addressed. Tel: 81-92-642-6615.
Fax: 81-92-642-6876.
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10.1021/jo051836t CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/25/2005
J. Org. Chem. 2006, 71, 125-134
125

Tanada et al.
binding motifs are located in close proximity to each other either
in a canonical duplex (Figure 1B) or in junction DNA structures
(Figure 1C). Here we wish to report in detail the molecular
design of new bidentate ligands, their synthesis. and their
selective binding to the DNA with nearby binding sites.
Results
Design of Bis-Hoechst Ligands. Hoechst 33258, a repre-
sentative minor-groove binder with high specificity to a
sequence of (A3T3)2,28-30 was chosen as a basic unit to construct
bidentate ligands (1). A polyethyleneoxy chain was used as a
linker by expecting flexibility in conformation and low affinity
with the phosphate backbone or base pairs. Introduction of the
linker at the convex side of the curvature of the Hoechst
skeletons would be advantageous in the point that the linker is
stretched out to the groove exterior over the phosphate backbone
to hold two minor groove binders within the complex with two
DNA duplexes as shown in Figure 2.
Synthesis of Bis-Hoechst Ligands. The carboxy-substituted
Hoechst derivatives were synthesized starting with 3,4-dini-
trobenzoic acid (2) and 3-amino-5-chloro-2-nitrobenzoic acid
(5) by modification of the reported method31 (Scheme 1). The
carboxylic acid of 2 was transformed to the Weinreb amide,
and the nitro groups were reduced to the corresponding diamine
(3). The diamino group was condensed with the p-hydroxyben-
zaldehyde, and the Weinreb amide was reduced to an aldehyde
group to yield the benzimidazole intermediate (4). The N-
methylpiperazine derivative (6) was obtained by esterification
and following substitution of chloride of 5.32 The nitro group
of 6 was reduced to the corresponding diamine, which was
coupled with 4, and then hydrolysis of the product gave the
Hoechst intermediate (7).
Synthesis of the bis-Hoechst ligands (1) was carried out by
solid-phase synthesis (Scheme 2). Fmoc-amino acids (17 and
18) were introduced to the resin, and the Fmoc protecting group
was removed to give the amino-terminal resin 8 or 11,
respectively. Each resin was subjected to further coupling with
18 for elongation of the linker. Finally, the carboxy-substituted
Hoechst intermediate 7 was applied to the resin, and the linker-
bearing Hoechst ligands (10, 12, and 13) were cleaved from
the resin with 10% TFA in CH2Cl2.
In the meantime, the anchor unit that is used to immobilize
the bis-Hoechst ligand onto the SPR sensor chip was synthesized
on the commercially available resin (14) with a diaminoether
linker unit. The anchor of 14 was elongated with 18 for two
cycles and then coupled with 19 to give the bis-amino terminal
(16). The linker-containing Hoechst intermediates (10, 12, and
13) were coupled with 16, and finally, the bis-Hoechst ligand
was cleaved from the resin with TFA. The mono-Hoechst (15)
as a reference compound having a long linker was also
FIGURE 1. General concept for the molecular design of the bidentate
ligand: (A) conceptual structure of the bidentate ligand; (B) cooperative
binding to nearby binding sites in a canonical duplex DNA; (C) possible
binding to distant sites in proximity of branched DNA.
cells.17 The expanded minor-groove binder has been shown to
form a complex with two remote binding sites at a distance of
approximately 70 base pairs to each other in nucleosomal
DNA.18 RNA functions are inhibited specifically by RNA-
binding ligands.19-22 Inspired by these successful examples, we
became interested in the development of small molecular ligands
for branched DNA junctions that are involved in a variety of
biological processes such as homologous recombination23 or
repair.24 There are a number of binding proteins for branched
DNA structures, but only a few studies have determined small
molecular binders.25-27 In this study, we have attempted to
establish a new design concept of small molecular ligands for
such higher-order DNA structure. Our approach relies on a gen-
eral structure of branched DNA that brings each arm into prox-
imity to the other and a bidentate ligand connecting two binder
structures with an appropriate linker (Figure 1). If two binding
domains of a bidentate ligand could interact with two remote
regions of DNA cooperatively, such a bidentate ligand would
show high affinity toward a specific DNA site where two distant
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I.; Mu¨ller, E.; Pascolo, E.; Sauter, G.; Pantic, M.; Martens, U. M.; Wenz,
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U.S.A. 2005, 102, 6867-6872.
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Nucleic Acids Res. 1997, 25, 4487-4492.
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C.; Rana, T. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12997-13002.
(23) A recent review; Yamada, K.; Ariyoshi, M.; Morikawa, K. Curr.
Opin. Struct. Biol. 2004, 14, 130-137.
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126 J. Org. Chem., Vol. 71, No. 1, 2006

Design of New Bidentate Ligands
FIGURE 2. Expected complex between two duplexes and the bis-Hoechst ligand (1) connected with a polyethyleneoxy linker stretched out to the
groove exterior.
SCHEME 1a
a (a) (1) SOCl2, 80 °C, 3 h, (2) CH3ONHCH3HCl, pyridine, rt, 3.5 h, 65%; (3) H2, 5% Pd-C, EtOH, rt; (b) (1) p-hydroxybenzaldehyde, Na2S2O5, EtOH,
H2O, reflux, 6 h, 50%; (2) LiAlH4, THF, -78 to 0 °C, 46 h, 84%; (c) (1) MeOH, H2SO4, reflux, 90 h, 74%, (2) N-methylpiperazine, K2CO3, 3.5 h, 52%;
(d) (1) H2, 5% Pd-C, EtOH, rt, 6.5 h, (2) 4, Na2S2O5, EtOH, H2O, reflux, 20 h, 94%, (3) 10% aqueous NaOH, 70 °C, 30 min, 84%.
synthesized similarly. The bis-Hoechst ligands (1) are named
according to the length of the linker: Short Bis-H (1S; m,n )
1,1), Middle Bis-H (1M; m,n ) 0,2), and Long Bis-H (1L; m,n
) 0,3). All products were purified by HPLC and their structures
were confirmed by MS, NMR measurements.
mono-Hoechst ligand 15, both association and dissociation of
DNA1 with a single A3T3 site are rapid. The binding constant
obtained here is much lower than that of the parent Hoechst
33258 measured by ITC (2.7 × 104 vs 3.2 × 108 M-1).30 It
was anticipated in a similar biding study by SPR that the small
ligand immobilized in the sensor surface would not be ef-
fectively exposed on the surface but would be folded back into
the matrix as the result of a small molecular size and its
hydrophobic character.33 Strong binding affinity of the mono-
Hoechst 15 to DNA1 in solution has been proved by CD titration
experiments as described in the subsequent section, indicating
that the decrease in binding affinity of the immobilized mono-
Hoechst 15 is probably due to such interfering factors. Figure
4A also indicates that DNA2 with two A3T3 sites binds to the
mono-Hoechst 15 stronger than DNA1 regardless of the distance
between the two A3T3 sites. As one molecule of the immobilized
ligand is estimated to exist in every 3-10 nm2, the neighboring
ligands on the sensor chip are thought to be close enough to
bind simultaneously two A3T3 sites of DNA2. Such a 2:1 ligand-
to-DNA complex has been also shown by CD titration experi-
ments using the free ligand 15 and DNA2(6) in the subsequent
section.
Evaluation of DNA Binding Properties. Surface Plasmon
Resonance (SPR). The Hoechst ligand containing the amino
terminal anchor unit (1, 15) was dissolved in acetate buffer (pH
4.5, 10 µM) and immobilized on a sensor chip CM5 (carboxym-
ethylated dextran surface) using amine coupling kit in HBS-N
running buffer (0.01 M HEPES, 0.15 M NaCl, pH 7.4) at 25
°C. The amounts of the immobilized ligand were monitored and
quantified by an increase of the SPR signal to be within the
range from 6.2 × 10-10 to 1.7 × 10-9 fmol/nm2, implying that
one ligand molecule is immobilized in every 3-10 nm2. DNA
samples (DNA1 and 2, Figure 3) were annealed prior to the
binding analysis by heating to 80 °C for 3 min and slowly cooled
to room temperature. SPR measurements were performed in
HBS-N running buffer at 25 °C. The reference response from
the blank cell was subtracted from the response in the channel
with the immobilized ligand to give a signal (RU, resonance
units) that is directly proportional to the amount of the bound
compound. SPR sensorgrams of the different DNA samples at
the same concentration for binding to the immobilized ligand
are compared in Figure 4. A set of sensorgrams at different
concentrations of the DNA samples (1 and 2(3)) for the mono-
and bis-Hoechst ligands (15, 1S, 1M, and 1L) were obtained
to calculate complex stability constants from Scatchard plots
(Table 1). As can be seen in the Figure 4A obtained with the
In striking contrast to the mono-Hoechst ligand 15, binding
affinity of the bis-Hoechst ligand (1) to DNA1 decreased
considerably (Figure 4B-D). Each bis-Hoechst ligand with a
linker of different length shows different SPR sensitivity
(33) Nakatani, K.; Kobori, A.; Kumasawa, H.; Saito, I. Bioorg. Med.
Chem. Lett. 2004, 14, 1105-1108.
J. Org. Chem, Vol. 71, No. 1, 2006 127

Tanada et al.
SCHEME 2. Solid-Phase Synthesis of Linker-Bearing Hoechst Ligandsa
a (a) Fmoc-â-alanine (17), DIPEA, CH2Cl2; (b) 20% piperidine, DMF; (c) 18, HBT, HBTU, DIPEA, NMP, DMF; (d) 7, BOP, HBT, DMF, NMP; (e) 10%
TFA in CH2Cl2; (f) 18, DIPEA, CH2Cl2; (g) 12, HBT, HBTU, DIPEA, NMP, DMF; (h) 19, HBT, HBTU, DIPEA, NMP, DMF; (i) 10, 12, or 13, HBT,
HBTU, DIPEA, NMP, DMF.
parameters in this study. We next examined DNA binding in
solution to determine specificity of the bis-Hoechst ligand. The
Long Bis-H (1L; m,n ) 0,3) was used for further investigation
in solution as a representative bis-Hoechst ligand, since it
discriminates the DNA substrates most effectively in the SPR
experiments.
Fluorescent Spectra Measurements. The fluorescent spectra
of mono-Hoechst ligand 15 showed high intensity even in the
absence of the DNA substrates (Figure 5A), and the DNA
substrates (1 and 2) were not discriminated. As shown in Figure
5B, fluorescent intensity of Long Bis-H (1; m,n ) 0,3) is
increased to a larger extent in the presence of DNA with two
A3T3 sites (DNA2) than in the presence of DNA1 with a single
A3T3 site, again demonstrating the selectivity of Long Bis-H
(1L; m,n ) 0,3) to the DNA substrates with two A3T3 sites.
Attempts to determine binding constants by these fluorescent
spectra changes were unsuccessful, because the spectral intensity
and maximum wavelength gradually changed during titration.
Circular Dichroism Spectra Measurements. It has been
reported that the binding of Hoechst 33258 to DNA is
accompanied by CD spectral changes between 200 and 300 nm
(the DNA region) and between 300 and 400 nm (a binding-
induced CD band of Hoechst 33258).34 CD titration profiles
were measured by adding aliquots of the Hoechst ligand to a
DNA solution. The ligand concentrations were in the range of
0-20 µM. Figure 6 summarizes the results with Long Bis-H
(1L; m,n ) 0,3), mono-Hoechst 15, and DNA1 or DNA2(6).
The CD spectrum of DNA2(6) was changed by the addition of
Long Bis-H (1L) with isoelliptic points at 257 and 284 nm
(Figure 6A), suggesting a single binding mode in relation to
FIGURE 3. Sequences of the duplexes used in this study. DNA1
contains a single A3T3 region, and DNA2(x) have two A3T3 regions
separated by x number of nonbinding base pairs.
according to the distance between the two A3T3 sites in the order
of DNA2(3) > DNA2(1), (6) > DNA2(9) . DNA1. Despite
differences in SPR sensitivity, calculated binding constants were
not very much different (Table 1). SPR binding experiments
have clearly demonstrated that the bis-Hoechst ligands selec-
tively bind the DNA substrates with two A3T3 sites compared
to the DNA substrate with a single A3T3 site.
As discussed in the analysis of the SPR sensorgrams obtained
with the mono-Hoechst ligand 15, sensorgrams with bis-Hoechst
ligands may contain effects by the nearby ligands immobilized
on the sensor chip. Decreased binding affinity of the mono-
Hoechst ligand 15 has also suggested that binding of the Hoechst
unit might be inhibited by its interaction with the matrix on the
sensor surface. Thus, it turned out that measurement of binding
affinity on the SPR sensor chip did not produce precise binding
(34) Han, F.; Taulier N.; Chalikian, T. V. Biochemistry 2005, 44, 9785-
9794.
128 J. Org. Chem., Vol. 71, No. 1, 2006

Design of New Bidentate Ligands
FIGURE 4. SPR measurement was performed with use of the sensor chip immobolizing Hoechst ligand (1 or 15). DNA duplexes (1, 2, 10 µM)
were injected during 0-120 s in HBS-N buffer, at pH 7.4, 25 °C.
TABLE 1. Complex Stability Constants Obtained from SPR
Measurementsa
DNA substrates are summarized in Table 2 and have clearly
demonstrated that Long Bis-H (1L; m,n ) 0,3) exhibits selective
binding to DNA2(6) with two A3T3 sites separated by six
nonbinding base pairs.
ligand
DNA
Ka (M-1
)
15
DNA1b
2.7 × 104
2.6 × 106
3.5 × 105
3.4 × 105
3.2 × 105
2.8 × 105
3.2 × 105
4.3 × 105
3.2 × 105
2.4 × 105
5.0 × 105
3.2 × 105
4.5 × 105
2.4 × 105
DNA2(3)c
DNA2(1)
DNA2(3)
DNA2(6)
DNA2(9)
DNA2(1)
DNA2(3)
DNA2(6)
DNA2(9)
DNA2(1)
DNA2(3)
DNA2(6)
DNA2(9)
Application of the Long Bis-Hoechst Ligand to Recogni-
tion of the Junction DNA. The results described above have
suggested that two Hoechst units of the Long Bis-H (1L; m,n
) 0,3) can bind two A3T3 sites simultaneously and discriminate
the distance between them. Therefore, we further examined
binding properties of Long Bis-H (1L, m,n ) 0,3) toward junc-
tion DNA structures as a model of highly ordered DNA. The
junction sequences were designed according to ref 35. The junc-
tion DNA3 and 4 were formed by mixing the three strands A,
B, and C (Figure 7), and its formation was analyzed by gel
electrophoresis with nondenatured polyacrylamide gel, followed
by isolation of the corresponding band and MALDI-TOF MS
measurements of the contained three strands. Junction DNA4(x,y)
having a single or two A3T3 sites was also synthesized using
the corresponding strands (A, B, and C). The positions of A3T3
sites in the junction DNA4(x,y) are named after the number of
bases x and y from the junction point, as shown in Figure
7.
The binding of Long Bis-H to the junction DNA 3 and 4
was analyzed by SPR (Figure 8A). Although DNA3 con-
tains no A3T3 site, weak binding with slow association and dis-
sociation rates was observed. A junction site may provide a
weak binding pocket to a Hoechst unit such as reported in
binding with the CC bulge site of RNA.36 Such additional
binding may result in relatively strong binding of Long Bis-H
(1L; m,n ) 0,3) with the junction DNA4(14, null) with a single
A3T3 site. This binding property shows a contrast to the nonbind-
ing ability observed with a straight duplex DNA1 (Figure 4D).
Short Bis-H
Middle Bis-H
Long Bis-H
a Complex stability constants were obtained from Scatchard plots except
for the data with ligand 15. The binding parameters with the ligand 15
were calculated by a built-in program using the following binding modes:
(b) Langmuir 1:1 binding, (c) bivalent 2:1 ligand-to-DNA binding.
the conformation of DNA2(6). Figure 6B represents a titration
curve obtained by plotting the changes of ellipticity at 275 nm,
and clearly indicates that complexation between Long Bis-H
(1; m,n ) 0,3) and DNA2(6) takes place in a 1:1 ratio with
high affinity of Ks ) 6.6 × 107 M-1. Long Bis-H (1L; m,n )
0,3) induced too small CD changes with DNA1 to calculate
the binding affinity. The results with SPR, UV, and CD have
clearly indicated that Long Bis-H (1L; m,n ) 0,3) has selective
affinity to DNA2 and not to DNA1. In similar CD titration
experiments, the titration curve obtained with use of mono-
Hoechst ligand 15 and DNA1 represents 1:1 ligand-to-DNA1
binding (Figure 6C), and that obtained with 15 and DNA2(6)
shows 2:1 ligand-to-DNA complexation (Figure 6D). Binding
constants of these complexes are similar to the reported value
of the parent Hoechst 33258 (Ks ) 3.2 × 108, M-1),30 indicating
that the Hoechst unit of 15 bind the two A3T3 sites of DNA2
independently. The binding constants of Long Bis-H with other
(35) Assenberg, R.; Weston, A.; Cardy, D. L.; Fox, K. R. Nucleic Acids
Res. 2002, 30, 5142-5150.
(36) Cho, J.; Rando, R. R. Nucleic Acids Res. 2000, 28, 2158-2168.
J. Org. Chem, Vol. 71, No. 1, 2006 129

Tanada et al.
FIGURE 5. Fluorescence spectra of mono-Hoechst (15) and Long Bis-H(1) changed in the absence and presence of DNA. Measurements were
performed using 100 nM each of the ligand and the DNA in the buffer at pH 7.4 with excitation wavelength at 370 nm.
FIGURE 6. CD spectra in the presence and absence of the Hoechst ligand: (A) CD spectra change of DNA2(6) in the presence of Long Bis-H;
(B-D) changes of ellipticity at 275 nm are plotted against the concentration of the ligand. CD spectra were measured using 5 µM DNA and the
ligand ranging from 0 to 20 µM.
TABLE 2. Binding Constants of the Long Bis-H (1L)
two A3T3 sites showed higher SPR sensitivity than to the
junction DNA4(14,null) with a single A3T3 site. However, the
position of A3T3 sites in the junction structure is not clearly
discriminated in the sensorgrams. In the CD titration experi-
ments, isoelliptic points are observed at 255 and 287 nm,
suggesting a single binding mode in relation to the junction
DNA structure. Small CD changes indicate that complexation
induces a little conformational change compared to the binding
with the linear DNA3 (Figures 6A vs 8B). From a titration curve
obtained by plotting the changes of ellipticity at 275 nm, the
binding parameter of 1:1 complexation with the binding constant
Ks ) 2.1 × 105 (M-1) was calculated. The two A3T3 sites of
DNA4(14,10) are apart in the sequence (24 base pairs), but
Ks (M-1
)
a
DNA1
b
DNA2(1)
DNA2(3)
DNA2(6)
DNA2(9)
1.2 × 105
3.0 × 105
6.6 × 107
2.2 × 106
a Calculated from the titration curve, such as shown in Figure 6C.
b Binding is too weak to obtain binding constant under the same conditions.
The junction DNA4 having two A3T3 sites exhibits higher
affinity compared to DNA4 with a single or no A3T3 binding
motif. Long Bis-H (1L) binds the junction DNA4(14,10) having
130 J. Org. Chem., Vol. 71, No. 1, 2006

Design of New Bidentate Ligands
FIGURE 9. Molecular modeling of the Long Bis-H. MD and MM
were performed with the AMBER force field.
FIGURE 7. General structures of the junction DNA3 and 4. Junction
DNA3 (control) represents the basic junction sequence having no A3T3
site. The numbers x and y in DNA4(x,y) represent the number of base
pairs from the junction point as shown in an example of DNA4(14,-
10). DNA4(14, null) has only a single A3T3 site at the position x ) 14.
should be close together in space in the junction conformation
to be bound by two Hoechst unit of Long Bis-H (1L).
Discussion
Selectivity to the DNA Duplexes with Two A3T3 Binding
Motifs. The CD titration experiments have clearly shown that
the Long Bis-H (1L) is highly selective to the DNA duplexes
with two A3T3 binding motifs. This property is interesting,
because the mono-Hoechst attaching a polyether linker retains
strong binding affinity to a single A3T3 site (Figures 4A, 6C).
The MD and MM calculations of the free Long Bis-H (1L)
have suggested a self-aggregated conformation, in which
Hoechst units are stacked to each other and the polyether linker
is coiled around itself (Figure 9). In the three binding studies
by SPR, fluorescence, and CD titration, the Bis-Hoechst 1 did
not show affinity to DNA1 with a single A3T3 site. From these
results, the possibility of binding of a stacked Hoechst dimer
with a single A3T3 site is excluded. Instead, such a stacked
conformation of the Long Bis-H (1L) may become disadvanta-
geous for binding with a A3T3 site. Thus, it may be reasonably
assumed that two Hoechst units of the unwound Bis-Hoechst 1
bind with two A3T3 sites of the DNA2 in such a way as
illustrated in Figure 10. The distances between the two Hoechst
units depend on the extent of unwinding of the linker of the
Long Bis-H (1L). The linker may be partially coiled in the
complex with DNA2 (Figure 10A), and it may be fully extended
in the complex with the junction DNA4 (Figure 10B). A search
FIGURE 10. Illustrations of the simulated complexes of the Long
Bis-H with the duplex DNA2(6) (A) and that with the junction DNA
4(14,10) (B). MD and MM were performed with AMBER 94 force
field.
for appropriate linkers will be a crucial step to determine more
specific ligands toward each DNA structure in further studies.
Conclusion
In conclusion, this study has revealed that new ligands
connecting two Hoechst units through a polyether linker show
specific affinity to DNA having two A3T3 motifs and that the
distance between the two A3T3 motifs can be recognized depend-
FIGURE 8. SPR Sensorgrams obtained with immobilized Long Bis-H (1L) (A) and CD spectra change of DNA4(14,10) in the presence of Long
Bis-H (1L) (B). SPR measurements were performed using 10 µM DNA in the HBS-N buffer at pH 7.4 at 25 °C, CD spectra were measured using
5 µM DNA and the ligand ranging from 0 to 20 µM.
J. Org. Chem, Vol. 71, No. 1, 2006 131

Tanada et al.
quenched by addition of H2O and acidified with 10% HCl. The
mixture was extracted with EtOAc. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The residue
was dissolved in EtOAc and extracted with 10% NaOH. The
aqueous layer was acidified with concentrated HCl and extracted
with EtOAc. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The crude product was
recrystallized from hexane/EtOAc to give 5 (10.3 g, 32%) as an
orange solid: mp 183-184 °C; IR (powder) cm-1 3489, 3377,
1707, 1609, 1572;1H NMR (400 MHz, DMSO-d6) δ 7.12 (d, J )
2.3 Hz, 1H), 7.06 (s, 2H), 6.75 (d, J ) 2.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 166.2, 145.2, 137.8, 133.1, 129.2, 118.9,
115.3.
3-Amino-5-(4-methylpiperazin-1-yl)-2-nitrobenzoic Acid Meth-
yl Ester (6). Under argon, to a solution of 5 (6.75 g, 31.2 mmol)
in absolute MeOH (200 mL) was added H2SO4 (2 mL), and the
mixture was heated at reflux. After 90 h, the reaction mixture was
neutralized with powdered Na2CO3. The mixture was diluted with
H2O and extracted with EtOAc. The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. Column chro-
matography (silica gel, hexane/EtOAc, 2/1 to 1/1) afforded 3-amino-
5-chloro-2-nitrobenzoic acid methyl ester (5.33 g, 74%) as a red
solid. The aqueous layer was acidified with 10% HCl and extracted
with EtOAc. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to recover 5 (1.19 g, 16%) as
a brown solid: mp 106-107 °C; IR (powder) cm-1 3464, 3352,
1717, 1570, 1290; 1H NMR (500 MHz, DMSO-d6) δ 6.90 (d, J )
2.3 Hz, 1H), 6.80 (d, J ) 2.3 Hz, 1H), 5.86 (br, 2H), 3.90 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 166.3, 144.4, 140.3, 132.8, 129.6,
119.4, 118.1, 53.3; HRMS (ESI) m/z calcd for C8H7ClN2O4Na ([M
+ Na]+) 252.9987, found 252.9983.
Under argon, to a solution of 3-amino-5-chloro-2-nitrobenzoic
acid methyl ester (100 mg, 0.43 mmol) in DMF (2 mL) was added
N-methylpiperazine (273 µL, 2.46 mmol) and potassium carbonate
(341 mg, 2.47 mmol), and the solution was heated at 105 °C. After
3.5 h, the reaction mixture was filtered and concentrated under
reduced pressure. Column chromatography (silica gel, CHCl3/
MeOH, 8/1 to 6/1) afforded 6 (66 mg, 52%) as a red solid: mp
160-161 °C; IR (powder) cm-1 3406, 3263, 1728, 1605, 1572;
1H NMR (270 MHz, CDCl3) δ 6.33 (d, J ) 2.6 Hz, 1H), 6.13 (br,
2H), 5.97 (d, J ) 2.6 Hz, 1H), 3.89 (s, 3H), 3.38 (t, J ) 5.1 Hz,
4H), 2.52 (t, J ) 5.1 Hz, 4H), 2.35 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 168.6, 153.8, 147.0, 124.3, 110.0, 106.5, 99.2, 54.4, 53.0,
46.7, 46.0; HRMS (ESI) m/z calcd for C13H19N4O4 ([M + H]+)
295.1401, found 295.1448.
2′-(4-Hydroxy-phenyl)-6-(4-methyl-piperazin-1-yl)-1H,3′H-
[2,5′]bibenzoimidazolyl-4-carboxylic Acid (7). A solution of 6
(500 mg, 1.07 mmol) in EtOH (25 mL) was hydrogenated over
5% Pd/C (500 mg) at room temperature. After 6.5 h, the reaction
mixture was filtered through a bed of Celite to remove the catalyst
and concentrated under reduced pressure to afford the corresponding
diamine. To a solution of the above product in EtOH (25 mL) was
added a solution of sodium pyrosulfite (168 mg, 0.88 mmol) in
H2O (0.9 mL) and 4 (607 mg, 2.55 mmol), and then the reaction
mixture was heated at reflux. After 20 h, the reaction mixture was
concentrated under reduced pressure. Column chromatography
(silica gel, diethyl ether/MeOH, 3/1 to 3/2) afforded the methyl
ester of 7 (768 mg, 94%) as a yellow solid: mp 269-270 °C; IR
(powder) cm-1 3500-2800, 1612, 1445; 1H NMR (400 MHz,
MeOH-d4) δ 8.28 (s, 1H), 7.98 (d, J ) 8.4 Hz, 1H), 7.96 (d, J )
8.8 Hz, 2H), 7.67 (d, J ) 8.4 Hz, 1H), 7.62 (d, J ) 2.3 Hz, 1H),
7.46 (d, J ) 2.1 Hz, 1H), 6.93 (d, J ) 8.8 Hz, 2H), 4.04 (s, 3H),
3.24 (t, J ) 5.0 Hz, 4H), 2.67 (t, J ) 4.9 Hz, 4H), 2.37 (s, 3H);
13C NMR (125 MHz, DMSO-d6) δ 165.8, 159.6, 154.5, 153.4,
145.3, 128.4, 123.3, 121.8, 120.5, 115.8, 114.7, 52.6, 52.2, 47.3,
42.2; HRMS (ESI) m/z calcd for C27H27N6O3 ([M + H]+) 483.2139,
found 483.2103.
ing on the length of the linker. As an interesting application of
the new ligand, binding to the junction DNA has been achieved.
As a first approach for recognition of highly ordered DNA
structure, this study has represented a reliable molecular basis
to design junction-specific DNA-binding molecules.
Experimental Section
N-Methoxy-N-methyl-3,4-diaminobenzamide (3). Under argon,
a mixture of thionyl chloride (25 mL) and 3,4-dinitrobenzoic acid
(2) (10 g, 47.1 mmol) was heated at 80 °C. After 3 h, the reaction
mixture was concentrated under reduced pressure. The residue was
subjected to toluene azeotrope to give the corresponding acid
chloride as a brown oil. Under argon, to a solution of the above
acid chloride in CH2Cl2 (50 mL) was added pyridine (6.9 mL, 85.3
mmol) and N,O-dimethylhydroxylamine hydrochloride (5.72 g, 58.6
mmol) at 0 °C, and then the mixture was stirred at room
temperature. After 3.5 h, the reaction mixture was diluted with CH2-
Cl2 and washed with H2O and brine. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The residue
was recrystallized from diethyl ether to give N-methoxy-N-methyl-
3,4-dinitrobenzamide (7.79 g, 65%) as a pale yellow solid: mp
1
95-96 °C; IR (powder) cm-1 1639, 1535, 1360; H NMR (270
MHz, CDCl3) δ 8.30 (d, J ) 1.6 Hz, 1H), 8.11 (dd, J ) 1.6, 8.3,
1H), 7.96 (d, J ) 8.3 Hz, 1H), 3.59 (s, 3H), 3.43 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 164.7, 143.4, 142.2, 139.0, 133.5, 125.4,
124.8, 61.7, 33.0.
A solution of the above product (6.37 g, 25 mmol) in EtOH
(200 mL) was hydrogenated over 5% Pd/C (4 g) for 4 h at room
temperature. The reaction mixture was used for the next reaction
without purification.
2-(4-Hydroxyphenyl)-3H-benzoimidazole-5-carbaldehyde (4).
A mixture of the above mixture, a solution of sodium pyrosulfite
(4.54 g, 23.9 mmol) in H2O (6 mL), and p-hydroxybenzaldehyde
(3.36 g, 27.5 mmol) was heated at reflux. After 6 h, the reaction
mixture was cooled to room temperature and filtered through a bed
of Celite to remove the catalyst. The filtrate was concentrated under
reduced pressure. The residue was washed with MeOH to give 2-(4-
hydroxyphenyl)-3H-benzoimidazole-5-carboxylic acid N-methoxy-
N-methylamide (3.69 g, 50%) as a beige powder: mp 281-282
°C; IR (powder) cm-1 3245, 1613, 1576, 1380; 1H NMR (400 MHz,
MeOH-d4) δ 10.00 (s, 0.5H), 9.99 (s, 0.5H), 8.00 (d, J ) 8.6 Hz,
2H), 7.86 (s, 0.5H), 7.75 (s, 0.5H), 7.62-7.42 (m, 3H), 6.91 (d, J
) 8.9 Hz, 2H), 3.61 (s, 3H), 3.39 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 169.8, 159.5, 153.7, 128.4, 127.4, 122.4, 120.7, 118.4,
117.6, 115.8, 110.5, 60.5, 33.7; HRMS (APCI) m/z calcd for
C16H16N3O3 ([M + H]+) 298.1186, found 298.1179.
Under argon, to a suspension of the above product (972 mg,
3.27 mmol) in THF/diethyl ether (3/1, 120 mL) was added LiAlH4
(380 mg, 13.3 mmol) at -40 °C, and then the reaction mixture
was stirred at 4 °C. After 46 h, the mixture was poured into saturated
NH4Cl/ice (1/1) and stirred for 10 min. The solution was extracted
with EtOAc, and the organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was triturated
with EtOAc/diethyl ether (1/1) to afford 4 (654 mg, 84%) as a pale
yellow powder: mp >300 °C; IR (powder) cm-1 3300-3100, 1611,
1286; 1H NMR (500 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.07-8.06
(m, 1H), 8.03 (d, J ) 8.9 Hz, 2H), 7.73-7.68 (m, 3H), 6.93 (d, J
) 8.7 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 192.5, 159.9,
159.2, 156.2, 152.0, 131.3, 130.9, 128.7, 128.2, 123.4, 123.0, 121.2,
120.4, 115.9, 115.7, 114.9; HRMS (APCI) m/z calcd for C14H11N2O2
([M + H]+) 239.0815, found 239.0809.
3-Amino-5-chloro-2-nitrobenzoic Acid (5). Under argon, to a
solution of potassium tert-butoxide (94 g, 838 mmol) and copper-
(II) acetate monohydrate (2.2 g, 12.1 mmol) in DMF (400 mL)
was added a solution of O-methylhydroxylamine hydrochloride (20
g, 239 mmol) and 3-chloro-6-nitrobenzoic acid (25 g, 124 mmol)
in DMF (400 mL) at 0 °C. After 3 h, the reaction mixture was
A solution of the above methyl ester of 7 (1 g, 2.07 mmol) in 1
M NaOH (33 mL) was heated at 70 °C. After 30 min, the reaction
132 J. Org. Chem., Vol. 71, No. 1, 2006

Design of New Bidentate Ligands
mixture was neutralized by 10% HCl and the resulting precipitate
was filtered. The obtained solid was dried under reduced pressure
over P2O5 to afford 7 (819 mg, 84%) as a yellow solid. Analytical
sample was obtained by reverse-phase HPLC (Nacalai Tesque
COSMOSIL 5C18-AR-300 20 mm × 250 mm; eluents H2O (A)
and CH3OH (B); gradient 0-20 min, 40-50% B in A + B; flow
rate 10 mL/min; detection 254 nm): mp 295-296 °C; IR (powder)
The reaction solvent was removed by filtration. The remaining resin
was washed three times each with NMP (4 mL) and CH2Cl2 (4
mL) and dried under reduced pressure to afford the corresponding
resin containing the expanded linker with the amino terminal as a
pale yellow resin.
General Procedure of Coupling with Hoechst-COOH (7) on
Resin. A mixture of the resin and Hoechst-COOH (7) (0.5 equiv
to the resin), DIPEA (5 equiv to the resin), HBT (5 equiv to the
resin), and BOP (5 equiv to the resin) in DMF/NMP (0.5 mL/0.5
mL) was shaken at room temperature for 5 h. The reaction solvents
were removed by filtration. The remaining resin was washed three
times each with NMP (3 mL) and CH2Cl2 (3 mL) and dried under
reduced pressure.
Synthesis of Branched Linker on Resin (16). The resin 14 was
subjected to elongation of the linker with use of 18 two times to
give the linker-elongated resin. A mixture of the above resin, 19
(10 equiv to the resin), DIPEA (10 equiv to the resin), HBT (10
equiv to the resin) and HBTU (10 equiv to the resin) in DMF/
NMP (1 mL/2 mL) was shaken at room temperature for 15 h. The
reaction solvents were removed by filtration. The remaining resin
was washed three times each with NMP (3 mL) and CH2Cl2 (3
mL) and dried under reduced pressure. The unreacted amino residue
was capped by using benzoic anhydride (10 equiv to the resin),
DIPEA (10 equiv to the resin) in CH2Cl2 (4 mL), and the resin
was washed and dried under reduced pressure. The above resin
was shaken in DMF (3.2 mL) containing piperidine (0.8 mL) at
room temperature for 30 min. The reaction solvents were removed
by filtration. The remaining resin was washed and dried under
reduced pressure to afford the diaminolinker-containing resin (16)
as pale yellow resin.
Coupling of Linker-Containing Hoechst Intermediates (10,
12, and 13) with Diaminolinker-Containing Resin (16). A mixture
of the diamino-resin 16, the linker-containing Hoechst intermediates
(10, 12, or 13) (4-25 equiv to 16), DIPEA (4-25 equiv to 16),
and a solution of HBT (25 equiv to 16) and HBTU (4-25 equiv to
16) in DMF/NMP (0.75 mL/0.75 mL) was shaken at room
temperature for 20 h. The reaction solvents were removed by
filtration. The remaining resin was washed three times each with
NMP (3 mL) and CH2Cl2 (3 mL) and dried under reduced pressure
to afford the corresponding coupled product.
Cleavage and Purification of Linker-Bearing Hoechst Deriva-
tives (1, 10, 12, and 13). The Hoechst-containing resin was treated
with 10% TFA in CH2Cl2 (4 mL) in a 10 mL glass vial for 90 min
at room temperature. The mixture was filtered and the remaining
resin was washed with THF (3 mL). The combined filtrates were
concentrated under reduced pressure. The crude product was
purified by reverse-phase HPLC (nacalai tesque COSMOSIL 5C18-
AR-II 10 mm × 250 mm; eluents; 0.5% (v/v) TFA in H2O (A)
and 0.5% (v/v) TFA in acetonitrile (B); gradient 0-20 min, 10-
40% B in A + B; flow rate 4 mL/min; detection 254 nm) to afford
the Hoechst ligand. (8.68 µmol, 20%) as a yellow solid. Overall
yield was determined by quantitative measurement of 1H NMR in
DMSO-d6 with 4 mM maleic acid as an internal standard.
Hoechst Intermediate 10. The Hoechst intermediate 10 was
obtained from the resin 9 (50 mg, 63 µmol) and Hoechst-COOH
(7) (14.6 mg, 31 µmol) as a yellow solid (9.3 µmol, 30%): IR
(powder) cm-1 3100-2800, 1667, 1464, 1429, 1298, 1187, 1114;
1H NMR (400 MHz, MeOH-d4) δ 8.50 (s, 1H), 8.36 (d, J ) 8.6
Hz, 1H), 8.05 (d, J ) 8.8 Hz, 2H), 7.88 (d, J ) 8.6 Hz, 1H), 7.74
(d, J ) 2.2 Hz, 1H), 7.36 (d, J ) 2.4 Hz, 1H), 7.09 (d, J ) 8.6 Hz,
2H), 3.83-3.77 (m, 12H), 3.71-3.74 (m, 4H), 3.35-3.34 (m, 2H),
3.23 (t, J ) 7.0 Hz, 2H), 3.01 (s, 3H), 2.33 (t, J ) 6.9 Hz, 2H);
HRMS (ESI) m/z calcd for C35H41N8O7 685.3093 ([M + H]+),
found 685.3044.
1
cm-1 3500-2800, 1531, 1443; H NMR (400 MHz, MeOH-d4) δ
8.30 (s, 1H), 8.01-7.97 (m, 3H), 7.69 (d, J ) 8.4 Hz, 1H), 7.65
(d, J ) 2.1 Hz, 1H), 7.31 (d, J ) 2.1 Hz, 1H), 6.94 (d, J ) 8.6 Hz,
2H), 3.26 (t, J ) 4.7 Hz, 4H), 2.69 (t, J ) 4.8 Hz, 4H), 2.38 (s,
3H); 13C NMR (125 MHz, DMSO-d6/D2O, 9/1) δ 166.5, 163.0,
151.9, 149.5, 148.4, 135.9, 135.6, 132.6, 130.7, 126.4, 125.7, 120.2,
118.5, 117.8, 117.5, 115.5, 113.6, 113.3, 107.7, 105.4, 53.6, 47.1,
43.7; HRMS (ESI) m/z calcd for C26H25N6O3 469.1983 ([M + H]+),
found 469.1962.
Bis[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyl]-
aminoacetic Acid (19). To a solution of FmocNHCH2CHO37 (1
g, 3.56 mmol) in MeOH (17 mL) and acetic acid (3 mL) were
added glycine (107 mg, 1.43 mmol) and sodium cyanoborohydride
(224 mg, 3.56 mmol) at room temperature. After 7 h, to the reaction
mixture was added diethyl ether (30 mL), and resulting precipitate
was filtered. The obtained solid was dissolved in 10% NaOH, and
the solution was acidified at pH 2 with 10% HCl. The aqueous
solution was extracted with EtOAc, and the organic layer was dried
over Na2SO4 and concentrated under reduced pressure to afford
19 (345 mg, 40%) as a white solid: mp 131-132 °C (dec); IR
(powder) cm-1 3500-3200, 3150-2990, 1701, 1533; 1H NMR (400
MHz, DMSO-d6) δ 7.86 (d, J ) 7.7 Hz, 4H), 7.65 (d, J ) 7.5 Hz,
4H), 7.39 (t, J ) 7.5 Hz, 4H), 7.29 (t, J ) 7.3 Hz, 4H), 7.18 (br,
2H), 4.27 (d, J ) 6.7 Hz, 4H), 4.20-4.18 (m, 2H), 3.06-3.05 (m,
4H), 2.67-2.66 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 157.1,
143.8, 141.3, 127.7, 127.0, 125.0, 120.0, 66.7, 62.2, 47.2, 43.4;
HRMS (ESI) m/z calcd for C36H36N3O6 606.2599 ([M + H]+),
found 606.2601.
Solid-Phase Synthesis. Loading of 18 onto the Resin (11) as
a General Procedure. Under argon, to the stirred solution of 18
(289 mg, 0.75 mmol) in CH2Cl2 (4 mL) was added DIPEA (0.52
mL, 3 mmol), 2-chlorotrityl chloride resin (1-1.5 mmol/g, 500 mg,
0.5-0.75 mmol) at room temperature. After 3 h, the reaction solvent
was removed by filtration. The remaining resin was washed with
CH2Cl2/MeOH/DIPEA (17/2/1, 4 mL), CH2Cl2 (4 mL), DMF (4
mL), CH2Cl2 (4 mL), three times each, and dried under reduced
pressure to afford the resin loading 18 (0.86 mmol/g) as pale yellow
resin. A mixture of the above resin and piperidine (0.8 mL) was
shaken in DMF (3.2 mL) for 30 min at room temperature, and the
solvents were removed by filtration. The remaining resin was
washed three times each with NMP (4 mL) and CH2Cl2 (4 mL)
and dried under reduced pressure to afford the amino-containing
resin 11 as a pale yellow resin. Unreacted chlorotrityl groups were
blocked by using hexylamine/THF (1/6, 1.2 mL), and the resin was
washed and dried in the same way as above.
Resin (8) and (14). The resins 8 and 14 were prepared from
2-chlorotrityl chloride resin with the use of Fmoc-â-alanine or 18,
respectively, by a similar method as described above.
General Procedure of Coupling with 18 on Resin. To a
solution of the 18 (5 equiv to the resin) in NMP (1 mL) was added
the resin, DIPEA (10 equiv to the resin), and a solution of HBT
(10 equiv to the resin) and HBTU (10 equiv to the resin) in DMF
(1 mL) at room temperature. The mixture was shaken at room
temperature for 2 h. The reaction solvents were removed by
filtration. The remaining resin was washed three times each with
NMP (3 mL) and CH2Cl2 (3 mL) and dried under reduced pressure
to afford the corresponding N-Fmoc-containing resin. The reaction
was monitored by checking the residual amino group with use of
ninhydrin. A mixture of the above resin and piperidine (0.8 mL)
in DMF (3.2 mL) was shaken for 30 min at room temperature.
Hoechst Intermediate 12. The Hoechst intermediate 12 was
obtained from the resin 11 (50 mg, 43 µmol) and Hoechst-COOH
(7) (10.1 mg, 22 µmol) as a yellow solid (7.6 µmol, 35%): IR
(powder) cm-1 3100-2800, 1669, 1463, 1429, 1300, 1191, 1114;
1H NMR (400 MHz, MeOH-d4) δ 8.43 (s, 1H), 8.32 (d, J ) 8.6
(37) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001-3003.
J. Org. Chem, Vol. 71, No. 1, 2006 133

Tanada et al.
Hz, 1H), 8.02 (d, J ) 8.8 Hz, 2H), 7.84 (d, J ) 8.5 Hz, 1H), 7.70
(d, J ) 2.1 Hz, 1H), 7.32 (d, J ) 2.1 Hz, 1H), 7.07 (d, J ) 8.6 Hz,
2H), 4.04 (s, 2H), 3.87 (s, 2H), 3.84-3.78 (m, 8H), 3.73-3.66
(m, 4H), 3.58-3.56 (m, 2H), 3.49-3.46 (m, 2H), 3.36-3.33 (m,
4H), 3.17 (t, J ) 5.7 Hz, 4H), 3.00 (s, 3H); HRMS (ESI) m/z calcd
for C38H47N8O9 759.3461 ([M + H]+), found 759.3506.
Hoechst Intermediate 13. The Hoechst intermediate 13 was
obtained from the resin 11 (100 mg, 86 µmol) and Hoechst-COOH
(7) (20.1 mg, 43 µmol) as a yellow solid (8.7 µmol, 20%): IR
(powder) cm-1 3100-2800, 1663, 1470, 1429, 1298, 1198, 1130;
1H NMR (400 MHz, MeOH-d4) δ 8.51 (s, 1H), 8.38 (d, J ) 8.5
Hz, 1H), 8.06 (d, J ) 8.8 Hz, 2H), 7.90 (d, J ) 8.5 Hz, 1H), 7.76
(d, J ) 2.1 Hz, 1H), 7.36 (d, J ) 2.1 Hz, 1H), 7.10 (d, J ) 8.8 Hz,
2H), 4.07 (s, 2H), 3.89 (s, 2H), 3.88 (s, 2H), 3.84-3.78 (m, 6H),
3.73-3.72 (m, 2H), 3.64-3.62 (m, 2H), 3.58-3.53 (m, 6H), 3.49-
3.48 (m, 6H), 3.39-3.34 (m, 6H), 3.20 (t, J ) 5.6 Hz, 4H), 3.02
(s, 3H); HRMS (ESI) m/z calcd for C44H59N9O12 452.7136 ([M +
2H]2+), found 452.7126; MALDI-TOF MS m/z calcd for ([M +
Na]+) C44H57N9O12Na 927.0, found 926.2.
3.96 (s, 2H), 3.94 (s, 2H), 3.91 (s, 4H), 3.89 (s, 4H), 3.86-3.76
(m, 12H), 3.67-3.66 (m, 4H), 3.63-3.36 (m, 54H), 3.23-3.16 (m,
8H), 3.11-3.09 (m, 8H), 3.00 (s, 6H); HRMS (ESI) m/z calcd for
C100H143N23O25 516.5151 ([M + 4H]4+), found 516.5191; MALDI-
TOF MS m/z calcd for C100H139N23O25 2063.3 ([M + H]+), found
2062.3.
Long Bis-Hoechst (1L; m,n ) 0,3). Long Bis-Hoechst was
obtained from the branched resin (16) (100 mg, 40 µmol) and
Hoechst intermediate 13 (183 µmol) as a yellow solid (1.95 µmol,
5%): 1H NMR (400 MHz, MeOH-d4); δ 8.33 (s, 2H), 8.24 (d, J )
8.5 Hz, 2H), 7.97 (d, J ) 8.5 Hz, 4H), 7.76 (d, J ) 8.2 Hz, 2H),
7.64-7.63 (m, 2H), 7.27-7.26 (m, 2H), 7.02 (d, J ) 8.2 Hz, 4H),
3.98-3.97 (m, 8H), 3.90-3.89 (m, 8H), 3.86-3.81 (m, 16H),
3.75-3.74 (m, 4H), 3.69-3.49 (m, 55H), 3.44-3.34 (m, 16H),
3.25-3.23 (m, 8H), 3.20-3.12 (m, 4H), 3.00 (s, 6H); HRMS (ESI)
m/z calcd for C112H165N25O31 589.0520 ([M + 4H]4+), found
589.0514; MALDI-TOF MS m/z calcd for C112H162N25O31 2354.6
([M + H]+), found 2352.7.
SPR Measurement. Loading of Hoechst Ligand onto a Sensor
Chip. The ligand was dissolved in acetate buffer (pH 4.5, 10 µM)
and immobilized on a sensor chip CM5 (carboxymethylated dextran
surface) using an amine coupling kit in HBS-N running buffer (0.01
M HEPES, 0.15 M NaCl, pH 7.4) at 25 °C. The amount of the
ligand immobilized on the surface was monitored as an increase
of the SPR signal. The density of the immobilized ligand on the
surface was calculated by the difference in SPR response before
and after immobilization, in which SPR response of 1000 RU is
equivalent to the change in surface concentration of 1 ng/mm2. One
flow channel was always left as a blank for reference. The remaining
carboxyl groups on the sensor surface were quenched with 35 µL
of 1 M ethanolamine, pH 8.5 with flow rate of 10 µL/min.
Procedure of SPR Binding Experiments. Prior to binding
experiments, DNA sample in a buffer was annealed by slow cool-
down to room temperature after heating at 80 °C for 3 min. SPR
measurement was performed in NBS-N running buffer at 25 °C
Binding was measured at 20 µL/min for 120 s and dissociation for
150 s. DNA samples bound on the sensor surface were removed
following each measurement by using glycine-NaOH buffer (pH
1.5) as regeneration solution at 30 µL/min for 30 s.
Monomer Hoechst Ligand (15). The monomer Hoechst ligand
(15) was obtained from the diaminoether-containing resin (14) (0.4
mmol/g, 100 mg, 0.04 mmol) and Hoechst-COOH (7) (18.7 mg,
0.04 mmol) as a yellow solid (8.78 µmol, 22%): 1H NMR (500
MHz, MeOH-d4) δ 8.51 (s, 1H), 8.38 (d, J ) 8.5 Hz, 1H), 8.06 (d,
J ) 8.8 Hz, 2H), 7.90 (d, J ) 8.5 Hz, 1H), 7.77 (d, J ) 2.4 Hz,
1H), 7.37 (d, J ) 2.1 Hz, 1H), 7.10 (d, J ) 8.8 Hz, 2H) 3.97 (s,
2H), 3.96 (s, 2H), 3.92 (s, 2H), 3.89 (s, 2H), 3.84-3.79 (m, 6H),
3.74-3.72 (m, 4H), 3.70-3.61 (m, 14H), 3.57-3.52 (m, 12H),
3.48-3.38 (m, 8H), 3.25-3.22 (m, 4H), 3.20-3.11 (m, 4H), 3.01
(s, 3H); HRMS (ESI) m/z calcd for C56H84N12O16 590.3056 ([M +
2H]2+), found 590.3025; MALDI-TOF MS m/z calcd for C56H83-
N12O16 1180.3 ([M + H]+), found 1180.9.
Short Bis-Hoechst (1S; m,n ) 1,1). Short Bis-Hoechst was
obtained from the branched resin (16) (40 mg, 16.3 µmol) and
Hoechst intermediate 11 (65 µmol) as a yellow solid (0.084 µmol,
1%): 1H NMR (500 MHz, MeOH-d4) δ 8.17 (s, 2H), 8.04 (d, J )
8.2 Hz, 2H), 7.89 (d, J ) 8.7 Hz, 4H), 7.60 (d, J ) 8.5 Hz, 2H),
7.58-7.57 (m, 2H), 7.19-7.18 (m, 2H), 6.94 (d, J ) 8.7 Hz, 4H),
3.96 (s, 2H), 3.93 (s, 2H), 3.90 (s, 4H), 3.86-3.81 (m, 16H), 3.75-
3.74 (m, 4H), 3.69-3.34 (m, 49H), 3.20 (t, 4H, J ) 6.9 Hz), 3.16-
3.15 (m, 2H), 3.00 (s, 6H), 2.29 (t, J ) 6.9 Hz, 4H); HRMS (ESI)
m/z calcd for C94H131N23O21 479.4967 ([M + 4H]4+), found
479.4933; MALDI-TOF MS m/z calcd for ([M + H]+) C94H128N23O21
1916.2, found 1915.4.
Acknowledgment. This work has been supported by a
Grant-in-Aid for Scientific Research (A) from Japan Society
for the Promotion of Science (JSPS), and CREST from Japan
Science and Technology Agency (JST).
Middle Bis-Hoechst (1M; m,n ) 0,2). Middle Bis-Hoechst was
obtained from the branched resin (16) (50 mg, 20 µmol) and
Hoechst intermediate 12 (485 µmol) as a yellow solid (0.8 µmol,
4%): 1H NMR (500 MHz, MeOH-d4) δ 8.25 (s, 2H), 8.10 (d, J )
7.8 Hz, 2H), 7.93 (d, J ) 8.7 Hz, 4H), 7.66 (d, J ) 8.0 Hz, 2H),
7.64-7.62 (m, 2H), 7.24-7.22 (m, 2H), 6.96 (d, J ) 8.5 Hz, 4H),
Supporting Information Available: 1H NMR and MS spectra
of Hoechst analogues in Schemes 1 and 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO051836T
134 J. Org. Chem., Vol. 71, No. 1, 2006